engleski

The role of Tyrosyl-DNA-phosphodiesterases in the repair of DNA-protein crosslinks in vivo

DNA protein crosslink (DPCs) is a type of DNA lesion which occurs when protein becomes irreversibly covalently bound to DNA. If not repaired DPCs interfere with all DNA transactions thus causing genomic instability which can lead to cancer, accelerated aging and neurodegeneration. The orchestration of DPC repair pathway is still unknown. Recently, it has been shown that metalloproteases Wss1 in yeast and SPRTN in mammals play central role in the repair of DPCs. These proteases cleave crosslinked proteins, thus leaving protein remnants of unknown size in the DNA backbone. While proteases act ubiquitously, cleaving wide variety of general DPCs, tyrosyl phosphodiesterases 1 and 2 (TDP1 and 2) play crucial role in the removal of enzymatic DPCs, topoisomerases 1 and 2 cleavage complexes. TDP1 has been shown to remove protein remnant of TOP1cc by separating DPC from DNA backbone through esterase activity, most probably after SPRTN mediated proteolytic cleavage of TOP1 crosslinked to DNA. TDP2 can act (a) downstream of SPRTN mediated proteolysis of TOP2cc or (b) remove TOP2cc with ZATT independently of SPRTN proteolysis. We aim to show function of TDPs in DPC removal in vivo in zebrafish model using CRISPR/Cas9 mediated mutagenesis of TDPs active site via knock-in technology and fluorescent reporter. We have identified zebrafish TDPs and compared them to human orthologs in regard to phylogenetics, synteny and mRNA and protein expression, while functional studies are under way. Our study will reveal contribution of TDP1 and 2 in DPC repair pathway on the organismal level.

DNA protein crosslinks ; Tdp1 ; Tdp2 ; zebrafish ; CRISPR

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